DNA topoisomerases are nuclear enzymes that regulate the conformational changes in DNA topology by catalyzing the breakage and rejoining of DNA strands during the normal cell cycle. They relieve torsional stress during replication and transcription.
Five human DNA topoisomerases have been identified and characterized: topoisomerase I (TOPI), topoisomerase IIα (TOPIIα), topoisomerase IIβ (TOPIIβ), topoisomerase IIIα (TOP3α), and topoisomerase IIIβ (TOP3β). TOPI reversibly cleaves a single strand in duplex DNA molecule, whereas TOPII breaks and rejoins both DNA strands. These reactions are believed to occur via transient reaction intermediates, known as “cleavable complexes,” where the enzymes (or enzyme subunits) form covalent bonds involving a tyrosine and the cleaved phosphodiester bond of the DNA substrate backbone.
To date, TOPI, TOPIIα, and TOPIIβ have been demonstrated to be important molecular targets for antitumor drugs (Gurrieri, C., et al., J Natl Cancer Inst, 2004. 96(4):269-79). During the past few years topoisomerases have become important chemotherapeutic targets for cancer treatment. Camptothecin (CPT) and its derivatives are reported to act specifically at the level of the TOPI-DNA complex and stimulate DNA cleavage. Other agents, such as β-lapachone, act by blocking the formation of the topoisomerase I-DNA complex. Several novel compounds have been developed that can target either TOPI or TOPIIα/IIβ-isoforms, or that can target all three types of topoisomerases.
As described above, TOPI catalyzes changes in DNA topology via the formation of a reversible enzyme-DNA cleavage complex. Anti-tumor drugs targeting TOPI, such as camptothecin (CPT) and its derivatives, lock the TOPI-DNA complex, resulting in cytotoxic DNA lesions that trigger cell cycle arrest and cell death.
CPT-mediated stabilization of TOPI-DNA complexes also induces TOPI proteasome-mediated degradation, which prevents topoisomerase inhibitor mediated cell death (Gambacorta, M., et al., Am J Pathol, 1996. 149(6): p. 2023-35 and Koken, M. H., et al., 10(7): p. 1315-24). In breast and colorectal cancer cell lines, there is a correlation between the extent of CPT-induced TOPI degradation and CPT resistance. For example, the breast cancer cell line ZR-75-1 is extremely sensitive to CPT and is completely defective in CPT-induced TOPI degradation, while the breast cancer cell line BT474 is insensitive to CPT and exhibits effective CPT-induced TOPI degradation (Zhang, P., et al., Int J Cancer, 2000. 85(5): p. 599-605). Supporting an essential role for ubiquitin-mediated degradation in the emergence of drug resistance, inhibiting proteasomes abolishes CPT-induced degradation of TOPI and selectively sensitized BT474 cells to CPT-induced cytotoxicity and apoptosis (Zhang, P., et al., Int J Cancer, 2000. 85(5): p. 599-605). Human TOPII isozymes, TOPIIα and TOPIIβ, are targeted in cancer cells by anthracyclines, such as doxorubicin, Teniposide (VM26, Alexis Corp.) and epipodophylotoxins, such as etoposide (VP-16)(Sigma, Israel).
TOPII inhibitors act in two diverse mechanisms. The first mechanism is similar to the TOPI inhibitors (e.g. VP-16) (Gurrieri, C., et al., J Natl Cancer Inst, 2004. 96(4): p. 269-79), while the second mechanism (e.g. ICRF-193) inhibits the catalytic activity of TOPII without trapping the covalent ternary complex (Yu, J. H., et al., Cancer Res., 2004. 64(3): p. 928-33 and Son, S. H., et al., Cancer Gene Ther, 2004. 5: p. 5). Most inhibitors of topoisomerase II block the ligation step, leading to stabilized cleavable complexes between DNA and the enzyme. Most enzyme inhibitors function by docking into the enzyme active site or nearby allosteric site to block the reaction of the normal substrate. Inhibition of the topoisomerase II involves two parts: the aromatic part of the inhibitor molecule intercalates between DNA base pairs while another more polar portion interacts with topoisomerase.
Because many topoisomerase II inhibitors (e.g., doxorubicin, and etoposide) act as poisons rather than as classical competitive inhibitors, their action is dependent upon the level of the enzyme in cells. Rapidly proliferating cells, which contain relatively higher levels of topoisomerase II, appear to be more sensitive to these agents. On the other hand, differentiated cells have relatively low topoisomerase II levels and are much more resistant to the action of these inhibitors.
Similarly to TOPI, the TOPII-DNA-Drug complex becomes susceptible to proteasome-mediated degradation contributing to the emergence of drug resistance (Le, X. F., et al., Oncogene, 1998. 16(14): p. 1839-49 and He, D., et al., Chin Med J (Engl), 2003. 116(9): p. 1394-8). Proteasome inhibition can circumvent solid tumor resistance to TOPII-directed drugs (He, D., et al., Cancer Res., 1997. 57(10): p. 1868-72). Physiological cell conditions, such as but not limited to glucose deprivation and hypoxia, play a role in solid tumor drug resistance (Melnick, A. and J. D. Licht, Blood, 1999. 93(10): p. 3167-215). These tumor-specific conditions cause decreases in TOPIIα levels, rendering cells resistant to TOPII-targeted drugs such as etoposide and doxorubicin (Piazza, F. C. Gurrieri, & P. P. Pandolfi, Oncogene, 2001. 20(49): p. 7216-22).
There remains a continuing need for developing approaches and compositions that are useful for enhancing the therapeutic effects of topoisomerase inhibitors.